Crystal forms of 2-[4-[(2,3,4-trimethoxyphenyl)methyl]piperazin-1-yl]ethyl pyridine-3-carboxylate

ABSTRACT

The invention provides polymorphs of a compound of Formula (X): 
     
       
         
         
             
             
         
       
     
     The invention also provided pharmaceutical compositions containing polymorphs of the compound and methods treating conditions in a subject by providing polymorphs of the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Provisional Patent Application No. 63/046,120, filed Jun. 30, 2020, thecontents of which are incorporated by reference.

FIELD OF THE INVENTION

The invention relates to crystallographic forms of2-[4-[(2,3,4-trimethoxyphenyl)methyl]piperazin-1-yl]ethylpyridine-3-carboxylate.

BACKGROUND

Heart disease is the leading cause of death worldwide, accounting for 15million deaths across the globe in 2015. In many forms of heart disease,decreased cardiac efficiency stems from changes in mitochondrial energymetabolism. Mitochondria are sub-cellular compartments in whichmetabolites derived from glucose and fatty acids are oxidized to producehigh-energy molecules. Increasing fatty acid oxidation in the heartdecreases glucose oxidation, and vice versa. Glucose oxidation is a moreefficient source of energy, but in certain types of heart disease, suchas heart failure, ischemic heart disease, and diabetic cardiomyopathies,fatty acid oxidation predominates in cardiac mitochondria. As a result,the pumping capacity of the heart is reduced.

CV-8972, which has the IUPAC name2-[4-[(2,3,4-trimethoxyphenyl)methyl]piperazin-1-yl]ethylpyridine-3-carboxylate and the following structure:

was recently identified as a promising therapeutic candidate fortreating or preventing cardiac conditions due to its pharmacokineticprofile.

SUMMARY

Provided herein are crystallographic forms of CV-8972 and compositionscontaining them. The invention recognizes that crystals of CV-8972 existin multiple polymorphic forms and that one polymorph, Form A, is themost stable under conditions of ambient temperature and relativehumidity. Therefore, Form A crystals of CV-8972 are useful for themanufacture of pharmaceutical compositions. For example, pharmaceuticalcompositions that contain the Form A polymorph do not require specialhandling during storage or distribution. In addition, such compositionsmay retain their efficacy better than compositions containing otherpolymorphs or mixtures of polymorphs. The invention also providesmethods of treating cardiac conditions in subject using CV-8972polymorphs, such as Form A.

In an aspect, the invention provides crystals comprising a polymorph ofa compound of Formula (X):

The polymorph may be Form A, Form B, Form C, Form D, or Form E.

The crystal may be substantially free of one or more other polymorphs.For example, the crystal may include a Form A polymorph and besubstantially free of polymorphs of Form B, Form C, Form D, and Form E.

The crystal may include a hydrochloride salt of the compound of Formula(X). The crystal may include the compound of Formula (X) and thehydrochloride ion in a defined stoichiometric ratio. The crystal mayinclude the compound and the hydrochloride ion in a 1:3 stoichiometricratio.

The crystal may include a hydrated form of the compound of Formula (X).The crystal may include a monohydrate form of the compound. The crystalmay include an anhydrous form of the compound.

In another aspect, the invention provides pharmaceutical compositionsthat include a polymorph of the compound of Formula (X).

The polymorph may be Form A, Form B, Form C, Form D, or Form E.

The composition may be substantially free of one or more otherpolymorphs. For example, the composition may include a Form A polymorphand be substantially free of polymorphs of Form B, Form C, Form D, andForm E.

The composition may include a hydrochloride salt of the compound ofFormula (X). The composition may include the compound of Formula (X) andthe hydrochloride ion in a defined stoichiometric ratio. The compositionmay include the compound and the hydrochloride ion in a 1:3stoichiometric ratio.

The composition may include a hydrated form of the compound of Formula(X). The composition may include a monohydrate form of the compound. Thecomposition may include an anhydrous form of the compound.

The composition may be formulated for any route or mode ofadministration. The composition may be formulated for buccal, dermal,enteral, intraarterial, intramuscular, intraocular, intravenous, nasal,oral, parenteral, pulmonary, rectal, subcutaneous, topical, ortransdermal administration. The composition may be formulated foradministration by injection or with or on an implantable medical device(e.g., stent or drug-eluting stent or balloon equivalents).

The composition may be formulated as a single unit dosage. Thecomposition may be formulated as divided dosages.

The composition may contain a defined dose of the compound. The dose maycontain from about 10 mg to about 2000 mg, from about 10 mg to about1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 600mg, from about 10 mg to about 400 mg, from about 10 mg to about 300 mg,from about 10 mg to about 200 mg, from about 25 mg to about 2000 mg,from about 25 mg to about 1000 mg, from about 25 mg to about 800 mg,from about 25 mg to about 600 mg, from about 25 mg to about 400 mg, fromabout 25 mg to about 300 mg, about 25 mg to about 200 mg, from about 50mg to about 2000 mg, from about 50 mg to about 1000 mg, from about 50 mgto about 800 mg, from about 50 mg to about 600 mg, from about 50 mg toabout 400 mg, from about 50 mg to about 300 mg, about 50 mg to about 200mg, from about 100 mg to about 2000 mg, from about 100 mg to about 1000mg, from about 100 mg to about 800 mg, from about 100 mg to about 600mg, from about 100 mg to about 400 mg, from about 100 mg to about 300mg, about 100 mg to about 200 mg, from about 200 mg to about 2000 mg,from about 200 mg to about 1000 mg, from about 200 mg to about 800 mg,from about 200 mg to about 600 mg, from about 200 mg to about 400 mg,from about 200 mg to about 300 mg, from about 300 mg to about 2000 mg,from about 300 mg to about 1000 mg, from about 300 mg to about 800 mg,from about 300 mg to about 600 mg, or from about 300 mg to about 400 mgof the compound. The dose may contain about 10 mg, about 25 mg, about 50mg, about 100 mg, about 200 mg, about 300 mg, or about 400 mg of thecompound.

The composition may contain a crystal of the compound of Formula (X).The crystal may have any of the properties described above in relationto crystals of the compound.

In another aspect, the invention provides methods of treating acondition in a subject by providing to a subject having, or at risk ofdeveloping, a condition a composition containing a therapeuticallyeffective amount of a polymorph of a compound of Formula (X).

The polymorph may be Form A, Form B, Form C, Form D, or Form E.

The composition may have any of the properties described above inrelation to compositions that include the compound of Formula (X),including crystals of the compound.

The composition may be provided by any suitable route or mode ofadministration. The composition may be provided buccally, dermally,enterally, intraarterially, intramuscularly, intraocularly,intravenously, nasally, orally, parenterally, pulmonarily, rectally,subcutaneously, topically, transdermally, by injection, or with or on animplantable medical device (e.g., stent or drug-eluting stent or balloonequivalents).

The composition may be provided as a single unit dosage. The compositionmay be provided as divided dosages.

The composition may be provided in one dose per day. The composition maybe provided in multiple doses per day. The composition may be providedin two, three, four, five, six, eight, or more doses per day.

The composition may contain a defined dose of the compound, such as anyof the doses described above.

The dose or doses may be provided for a defined period. One or moredoses may be provided daily for at least one week, at least two weeks,at least three weeks, at least four weeks, at least six weeks, at leasteight weeks, at least ten weeks, at least twelve weeks or more.

The condition may be a cardiovascular condition. The cardiovascularcondition may be aneurysm, angina, atherosclerosis, cardiomyopathy,cerebral vascular disease, congenital heart disease, coronary arterydisease, coronary heart disease, diabetic cardiomyopathy, heart attack,heart disease, heart failure, hypertension, ischemic heart disease,pericardial disease, peripheral arterial disease, rheumatic heartdisease, stroke, transient ischemic attacks, or valvular heart disease.The angina may be refractory to other medical interventions.

The condition may be a rheumatic condition. The rheumatic condition maybe acute kidney injury, alcoholic cardiomyopathy, angina (e.g.,refractory angina and angina associated with heart failure), ankylosingspondylitis, autoimmune-related lung disease, Behcet's Disease,bursitis, cachexia, cardiac fibrosis, chemotherapy chronic fatiguesyndrome, claudication (e.g., peripheral claudication), contrastnephropathy, cyanotic heart disease, dermatomyositis, dilatedcardiomyopathy, disequilibrium, fibromyalgia, frailty, gout, Gulf Warsyndrome, heart failure, hypertrophic cardiomyopathy, inducednephropathy, infectious arthritis, inflammatory arthritis, inflammatoryeye disease, inflammatory myositis, ischemic cardiomyopathy, juvenileidiopathic arthritis, left ventricular dysfunction, lupus, musclemyopathy, myofascial pain syndrome, myositis, osteoarthritis,osteonecrosis of the jaw, osteoporosis, polymyalgia rheumatica,polymyositis, psoriatic arthritis, pulmonary arterial hypertension,pulmonary fibrosis, a rare muscle disease, rheumatoid arthritis,sarcoidosis, sarcopenia, scleroderma, Sjogren's syndrome, tendinitis,tinnitus, vasculitis, or vertigo.

The condition may fibrosis. The fibrosis may be associated with anotherdisease, disorder, or condition. For example, the fibrosis may includeor be associated with adhesive capsulitis, aneurysm, angina, arterialstiffness, arthrofibrosis, atherosclerosis, atrial fibrosis,cardiomyopathy, cerebral vascular disease, cirrhosis, congenital heartdisease. coronary artery disease, coronary heart disease, Crohn'sdisease, cystic fibrosis, diabetic cardiomyopathy, Dupuytren'scontracture, endomyocardial fibrosis, glial scar, heart attack, heartfailure, high blood pressure (hypertension), idiopathic pulmonaryfibrosis, ischemic heart disease, keloid, mediastinal fibrosis,myelofibrosis, nephrogenic systemic fibrosis, old myocardial infarction,pericardial disease, peripheral arterial disease, Peyronie's disease,progressive massive fibrosis, pulmonary fibrosis, radiation-induced lunginjury, retroperitoneal fibrosis, rheumatic heart disease, scleroderma,stroke, systemic sclerosis transient ischemic attacks, or valvular heartdisease.

The condition may be cancer. The cancer may be bladder cancer, braincancer, breast cancer, carcinoma, cervical cancer, colon cancer,colorectal cancer, gastric cancer, glioblastoma, glioma, head and neckcancer, kidney cancer, leukemia, liposarcoma, liver cancer, lung cancer,lymphoma, medullablastoma, melanoma, muscle cancer, neuroblastoma,oligoastrocytoma, oligodendroglioma, osteosarcoma, ovarian cancer,pancreatic cancer, paraganglioma, prostate cancer, sarcoma, or thyroidcancer.

In another aspect, the invention provides methods of altering cardiacremodeling by providing to a subject that has developed, or is at riskof developing, cardiac remodeling a composition containing atherapeutically effective amount of a polymorph of a compound of Formula(X).

The polymorph may be Form A, Form B, Form C, Form D, or Form E.

The composition may have any of the properties described above inrelation to compositions that include the compound of Formula (X),including crystals of the compound.

The composition may be provided by any suitable route or mode ofadministration. The composition may be provided buccally, dermally,enterally, intraocular intravenously, nasally, orally, parenterally,pulmonarily, subcutaneously, topically, transdermally, by injection, orwith or on an implantable medical device (e.g., stent or drug-elutingstent or balloon equivalents).

The composition may be provided as a single unit dosage. The compositionmay be provided as divided dosages.

The composition may be provided in one dose per day. The composition maybe provided in multiple doses per day. The composition may be providedin two, three, four, five, six, eight, or more doses per day.

The composition may contain a defined dose of the compound, such as anyof the doses described above.

The dose or doses may be provided for a defined period. One or moredoses may be provided daily for at least one week, at least two weeks,at least three weeks, at least four weeks, at least six weeks, at leasteight weeks, at least ten weeks, at least twelve weeks or more.

The cardiac remodeling may be associated with a disease, disorder, orcondition. The cardiac remodeling may be associated with acardiovascular disease. For example, the cardiac remodeling may beassociated with aberrant subclavian artery, aortic regurgitation, aorticstenosis, arteriovenous malformation and fistula, atrial septal defect,atrioventricular septal defect, bicuspid aortic valve, cardiomegaly,cardiomyopathy, coarctation of the aorta, complete heart block,concentric hypertrophy, congenital heart defects, congenital heartdisease, coronary artery disease, dextrocardia, dextro-transposition ofthe great arteries, diabetes, diet, double aortic arch, double inletleft ventricle, double outlet right ventricle, Ebstein's anomaly, gianthepatic hemangioma, heart failure, high cholesterol, high-outputhemodialysis fistula, hypertension, hypertension, hypoplastic left heartsyndrome, hypoplastic right heart syndrome, interrupted aortic arch,levo-transposition of the great arteries, mitral regurgitation, alsocausing left atrial volume overload, mitral stenosis, myocardialischemia, obesity, outflow obstruction, partial anomalous pulmonaryvenous connection, patent ductus arteriosus, pentalogy of Cantrell,persistent truncus arteriosus, pressure overload, pulmonary atresia,pulmonary hypertension, pulmonary regurgitation, pulmonary stenosis,rhabdomyomas, right ventricular volume overload, scimitar syndrome,Shone's syndrome, tetralogy of Fallot, total anomalous pulmonary venousconnection, transposition of the great vessels, tricuspid atresia,tricuspid regurgitation, use of tobacco, alcohol, or other drugs,valvular heart disease, ventricular dilation, ventricular hypertrophy,ventricular septal defect, volume overload, and Wolff-Parkinson-Whitesyndrome.

In another aspect, the invention provides uses of crystals containing apolymorph of a compound of Formula (X) for making a medicament.

In embodiments of the use, the polymorph is Form A, Form B, Form C, FormD, or Form E.

In embodiments of the use, the crystal is substantially free of one ormore other polymorphs. In embodiments of the use, the crystal includes aForm A polymorph and is substantially free of polymorphs of Form B, FormC, Form D, and Form E.

In embodiments of the use, the crystal includes a hydrochloride salt ofthe compound of Formula (X). In embodiments of the use, the crystalincludes the compound of Formula (X) and the chloride ion in a definedstoichiometric ratio. In embodiments of the use, the crystal includesthe compound and the chloride ion in a 1:3 stoichiometric ratio.

In embodiments of the use, the medicament includes a hydrated form ofthe compound of Formula (X). In embodiments of the use, the medicamentincludes a monohydrate form of the compound. In embodiments of the use,the medicament includes an anhydrous form of the compound.

In embodiments of the use, the medicament is formulated for buccal,dermal, enteral, intraarterial, intramuscular, intraocular, intravenous,nasal, oral, parenteral, pulmonary, rectal, subcutaneous, topical, ortransdermal administration. In embodiments of the use, the medicament isformulated for administration by injection or with or on an implantablemedical device (e.g., stent or drug-eluting stent or balloonequivalents).

In embodiments of the use, the medicament is formulated as a single unitdosage. In embodiments of the use, the medicament is formulated asdivided dosages.

In embodiments of the use, the medicament contains from about 10 mg toabout 2000 mg, from about 10 mg to about 1000 mg, from about 10 mg toabout 800 mg, from about 10 mg to about 600 mg, from about 10 mg toabout 400 mg, from about 10 mg to about 300 mg, from about 10 mg toabout 200 mg, from about 25 mg to about 2000 mg, from about 25 mg toabout 1000 mg, from about 25 mg to about 800 mg, from about 25 mg toabout 600 mg, from about 25 mg to about 400 mg, from about 25 mg toabout 300 mg, about 25 mg to about 200 mg, from about 50 mg to about2000 mg, from about 50 mg to about 1000 mg, from about 50 mg to about800 mg, from about 50 mg to about 600 mg, from about 50 mg to about 400mg, from about 50 mg to about 300 mg, about 50 mg to about 200 mg, fromabout 100 mg to about 2000 mg, from about 100 mg to about 1000 mg, fromabout 100 mg to about 800 mg, from about 100 mg to about 600 mg, fromabout 100 mg to about 400 mg, from about 100 mg to about 300 mg, about100 mg to about 200 mg, from about 200 mg to about 2000 mg, from about200 mg to about 1000 mg, from about 200 mg to about 800 mg, from about200 mg to about 600 mg, from about 200 mg to about 400 mg, from about200 mg to about 300 mg, from about 300 mg to about 2000 mg, from about300 mg to about 1000 mg, from about 300 mg to about 800 mg, from about300 mg to about 600 mg, or from about 300 mg to about 400 mg of thecompound. In embodiments of the use, the medicament contains about 10mg, about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg,or about 400 mg of the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a space-filling three-dimensional model of the crystalstructure of the Form D polymorph of CV-8972.

FIG. 2 is a space-filling three-dimensional model of the crystalstructure of the Form D polymorph of CV-8972 at room temperature.

FIG. 3 is a space-filling three-dimensional model of the crystalstructure of the Form A polymorph of CV-8972.

FIG. 4 is an XRPD diffractogram of the CV-8972 starting material.

FIG. 5 shows TGA and DSC thermograms of the CV-8972 starting material.

FIG. 6 shows XRPD diffractograms of various forms of CV-8972.

FIG. 7 is a polarized microscopic image of CV-8972 starting material.

FIG. 8 is a dynamic vapor sorption isotherm plot.

FIG. 9 shows XRPD diffractograms of CV-8972 before and after dynamicvapor sorption.

FIG. 10 shows XRPD diffractograms of CV-8972 in its dehydrated andrehydrated forms.

FIG. 11 shows XRPD diffractograms of various polymorphs of CV-8972.

FIG. 12 is a PLM image of a batch of single crystals of C₂₂H₃₄Cl₃N₃O₆(CV-8972).

FIG. 13 shows PLM images of a crystal used for single-crystaldiffractometer.

FIG. 14 shows images of a crystal mounted on a 100 micro Mitegen loop onthe diffractometer.

FIG. 15 is an Ortep diagram of an asymmetric unit of the C₂₂H₃₄Cl₃N₃O₆crystal.

FIG. 16 shows one unit cell of the C₂₂H₃₄Cl₃N₃O₆ crystal.

FIG. 17 is a diagram of hydrogen bonds networks and counter-ion pairs inthe C₂₂H₃₄Cl₃N₃O₆ crystal.

FIG. 18 shows calculated and measured XRPD diagrams of the C₂₂H₃₄Cl₃N₃O₆crystal.

FIG. 19 shows PLM images of single anhydrous crystals fromrecrystallized CV-8972.

FIG. 20 is an image of a single anhydrous crystal from recrystallizedCV-8972 mounted on a tip of a glass fiber.

FIG. 21 is a thermal ellipsoid diagram of an asymmetric unit of theC₂₂H₃₂C₁₃N₃O₅ crystal.

FIG. 22 shows one unit cell of the C₂₂H₃₂C₁₃N₃O₅ crystal.

FIG. 23 is a diagram of hydrogen bonds networks and counter-ion pairs inthe C₂₂H₃₂C₁₃N₃O₅ crystal.

FIG. 24 shows calculated and measured XRPD diagrams of the C₂₂H₃₄Cl₃N₃O₆crystal.

DETAILED DESCRIPTION

The recently-identified compound CV-8972 holds promise as a therapeuticagent for treating a variety of conditions, including cardiovascularconditions, rheumatic diseases, fibrosis, and cancer. CV-8972, which hasthe IUPAC name 2-[4-[(2,3,4-trimethoxyphenyl)methyl]piperazin-1-yl]ethylpyridine-3-carboxylate and the following structure:

is metabolized in the body into two sets of products that increasemitochondrial energy production in different ways. In an initialreaction, the molecule is split into CV-8814, which has the followingstructure:

and nicotinic acid. Over time, CV-8814 converted in the body totrimetazidine. Both CV-8814 and trimetazidine inhibit beta-oxidation offatty acids and therefore shift mitochondrial metabolism towardoxidation of glucose, a more oxygen-efficient source of energy.Nicotinic acid serves as precursor for synthesis of nicotinamide adeninedinucleotide (NAD⁺). NAD⁺ promotes mitochondrial respiration to driveATP synthesis, regardless of whether glucose or fatty acids are used asthe carbon source. Thus, the two sets of products that result frombreakdown of CV-8972 in vivo act synergistically to stimulate energyproduction in mitochondria in cardiac tissue and other cell types.CV-8972 and its mechanism of action are described in U.S. Pat. No.10,556,013, the contents of which are incorporated herein by reference.

The present invention recognizes that crystals of CV-8972 exist inmultiple polymorphic forms. One polymorph, Form A, is most stable underconditions of ambient temperature and relative humidity and thereforehas particular utility for the manufacture of pharmaceuticalcompositions. Due to the stability of Form A, compositions containingthis polymorph can readily be stored and distributed without loss oftherapeutic efficacy. Thus, the invention provides compositionscontaining polymorphs of crystalline CV-8972, methods of making suchcompositions, and methods of using them to treat various conditions in asubject.

Polymorphs of CV-8972

As described in the examples below, crystals of CV-8972 may exist in atleast five polymorphic forms: Form A, Form B, Form C, Form D, and FormE. Form A is monohydrate, and Forms B, D, and E are anhydrous. Form Cwas not obtained in purified form, so its hydration state could not bedetermined.

Crystals may be formed as salts of CV-8972. For example, crystals may beformed as hydrochloride salts of CV-8972.

FIG. 1 is a space-filling three-dimensional model of the crystalstructure of the Form D polymorph of CV-8972. The polymorph is atrihydrochloride salt, and chloride ions are shown in green.

FIG. 2 is a space-filling three-dimensional model of the crystalstructure of the Form D polymorph of CV-8972 at room temperature. Thepolymorph is a trihydrochloride salt, and chloride ions are shown ingreen.

FIG. 3 is a space-filling three-dimensional model of the crystalstructure of the Form A polymorph of CV-8972. The polymorph is atrihydrochloride salt, and chloride ions are shown in green.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions that contain crystalsof a polymorph of CV-8972. For example, the composition may containCV-8972 crystals in Form A, Form B, Form C, Form D, or Form E. Thecomposition may be substantially free of one or more other polymorphs.For example, the composition may include a Form A polymorph and besubstantially free of polymorphs of Form B, Form C, Form D, and Form E.

A composition containing a polymorph of CV-8972 may be substantiallyfree of one or more other polymorphic forms of CV-8972 if thecomposition contains the predominant polymorph at a defined level ofpurity. Purity may be expressed as the amount of predominant polymorphas a percentage of the total weight of two of more polymorphs ofCV-8972.

In certain embodiments, the total weight is the weight of all polymorphsof CV-8972 in the composition. For example, a composition that containsthe Form A polymorph and is substantially free of other polymorphs maycontain Form A at a defined weight percentage of all polymorphs ofCV-8972 in the composition. For example, the composition may containForm A at at least 95% by weight, at least 96% by weight, at least 97%by weight, at least 98% by weight, at least 99% by weight, at least99.5% by weight, at least 99.6% by weight, at least 99.7% by weight, atleast 99.8% by weight, or at least 99.9% by weight of all polymorphs ofCV-8972 in the composition.

In certain embodiments, the total weight is the weight of selectedpolymorphs of CV-8972 in the composition. For example, a compositionthat contains the Form A polymorph and is substantially free of the FormB polymorph may contain Form A at a defined weight percentage of Forms Aand B. For example, the composition may contain Form A at at least 95%by weight, at least 96% by weight, at least 97% by weight, at least 98%by weight, at least 99% by weight, at least 99.5% by weight, at least99.6% by weight, at least 99.7% by weight, at least 99.8% by weight, orat least 99.9% by weight of Forms A and B of CV-8972 in the composition.Similarly, a composition that contains the Form A polymorph and issubstantially free of the Form B and C polymorphs may contain Form A ata defined weight percentage of Forms A, B, and C. For example, thecomposition may contain Form A at at least 95% by weight, at least 96%by weight, at least 97% by weight, at least 98% by weight, at least 99%by weight, at least 99.5% by weight, at least 99.6% by weight, at least99.7% by weight, at least 99.8% by weight, or at least 99.9% by weightof Forms A, B, and C of CV-8972 in the composition.

Alternatively or additionally, a composition containing a polymorph ofCV-8972 may be substantially free of one or more other polymorphic formsof CV-8972 if the composition contains the secondary polymorphs atlevels below a defined level. Presence of a secondary polymorphs may bedefined as the amount of one or more secondary polymorphs as apercentage of the total weight of two of more polymorphs of CV-8972.

In certain embodiments, the total weight is the weight of all polymorphsof CV-8972 in the composition. For example, a composition that containsthe Form A polymorph and is substantially free of other polymorphs maycontain all polymorphs other than Form A at a defined weight percentageof all polymorphs of CV-8972 in the composition. For example, thecomposition may contain all polymorphs other than Form A at below 5% byweight, below 4% by weight, below 3% by weight, below 2% by weight,below 1% by weight, below 0.5% by weight, below 0.4% by weight, below0.3% by weight, below 0.2% by weight, or below 0.1% by weight of allpolymorphs of CV-8972 in the composition.

In certain embodiments, the total weight is the weight of selectedpolymorphs of CV-8972 in the composition. For example, a compositionthat contains the Form A polymorph and is substantially free of the FormB polymorph may contain Form B at a defined weight percentage of Forms Aand B. For example, the composition may contain Form B at below 5% byweight, below 4% by weight, below 3% by weight, below 2% by weight,below 1% by weight, below 0.5% by weight, below 0.4% by weight, below0.3% by weight, below 0.2% by weight, or below 0.1% by weight of Forms Aand B of CV-8972 in the composition. Similarly, a composition thatcontains the Form A polymorph and is substantially free of the Form Band Form C polymorphs may contain Forms B and C at a defined weightpercentage of Forms A, B, and C. For example, the composition maycontain Forms B and C at below 5% by weight, below 4% by weight, below3% by weight, below 2% by weight, below 1% by weight, below 0.5% byweight, below 0.4% by weight, below 0.3% by weight, below 0.2% byweight, or below 0.1% by weight of Forms A, B, and C of CV-8972 in thecomposition.

The composition may include a hydrochloride salt of a CV-8972 polymorph.The composition may include CV-8972 and the chloride ion a definedstoichiometric ratio. The composition may include CV-8972 and thechloride ion in a 1:3 stoichiometric ratio.

The composition may include a hydrated form of CV-8972. The compositionmay include a monohydrate form of CV-8972, such as the Form A polymorph.The composition may include an anhydrous form of CV-8972, such as a FormB, Form D, or Form E polymorph.

The composition may be formulated for any route or mode ofadministration. The composition may be formulated for buccal, dermal,enteral, intraarterial, intramuscular, intraocular, intravenous, nasal,oral, parenteral, pulmonary, rectal, subcutaneous, topical, ortransdermal administration. The composition may be formulated foradministration by injection or with or on an implantable medical device(e.g., stent or drug-eluting stent or balloon equivalents).

The composition may be formulated as a single unit dosage. Thecomposition may be formulated as divided dosages.

The composition may contain a defined dose of CV-8972. The dose maycontain from about 10 mg to about 2000 mg, from about 10 mg to about1000 mg, from about 10 mg to about 800 mg, from about 10 mg to about 600mg, from about 10 mg to about 400 mg, from about 10 mg to about 300 mg,from about 10 mg to about 200 mg, from about 25 mg to about 2000 mg,from about 25 mg to about 1000 mg, from about 25 mg to about 800 mg,from about 25 mg to about 600 mg, from about 25 mg to about 400 mg, fromabout 25 mg to about 300 mg, about 25 mg to about 200 mg, from about 50mg to about 2000 mg, from about 50 mg to about 1000 mg, from about 50 mgto about 800 mg, from about 50 mg to about 600 mg, from about 50 mg toabout 400 mg, from about 50 mg to about 300 mg, about 50 mg to about 200mg, from about 100 mg to about 2000 mg, from about 100 mg to about 1000mg, from about 100 mg to about 800 mg, from about 100 mg to about 600mg, from about 100 mg to about 400 mg, from about 100 mg to about 300mg, about 100 mg to about 200 mg, from about 200 mg to about 2000 mg,from about 200 mg to about 1000 mg, from about 200 mg to about 800 mg,from about 200 mg to about 600 mg, from about 200 mg to about 400 mg,from about 200 mg to about 300 mg, from about 300 mg to about 2000 mg,from about 300 mg to about 1000 mg, from about 300 mg to about 800 mg,from about 300 mg to about 600 mg, or from about 300 mg to about 400 mgof CV-8972. The dose may contain about 10 mg, about 25 mg, about 50 mg,about 100 mg, about 200 mg, about 300 mg, or about 400 mg of CV-8972.

A pharmaceutical composition containing a polymorph of CV-8972 may be ina form suitable for oral use, such as tablets, troches, lozenges,fast-melts, dispersible powders or granules, or capsules. Compositionsintended for oral use may be prepared according to any method known inthe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from sweeteningagents, flavoring agents, coloring agents and preserving agents, inorder to provide pharmaceutically elegant and palatable preparations.Tablets contain the polymorph in admixture with non-toxicpharmaceutically acceptable excipients. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example corn starch, or alginic acid; bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid, or talc. Preparation andadministration of pharmaceutical compositions is discussed in U.S. Pat.No. 6,214,841 and U.S. Patent Publication No. 2003/0232877, the contentsof each of which are incorporated by reference herein. Formulations fororal use may also be presented as hard gelatin capsules in which thecompounds are mixed with an inert solid diluent, such as calciumcarbonate, calcium phosphate or kaolin. The formulation may allowcontrolled release of the polymorph of CV-8972 in the gastrointestinaltract by encapsulating the polymorph in an enteric coating.

Dispersible powders and granules provide the compounds in admixture witha dispersing or wetting agent, suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may contain mixtures that include erodiblepolymers that promote swelling of the mixture in an aqueous environment.An erodible polymer is any polymer that breaks down inside the bodywithin a physiologically relevant time frame. The erodible polymer mayhave other characteristics that promote the gradual release of thepolymorphic form of CV-8972 from the mixture. For example and withoutlimitation, the polymer may be one or more of the following:biocompatible, i.e., not harmful to living tissue; hydrophilic;hygroscopic; tending to form a hydrogel.

Without wishing to be bound by theory, the polymer-containing mixturesmay promote gradual release by one or more mechanisms. For example,swelling of the mixture by absorption of water may facilitate diffusionof the polymorphic form of CV-8972 from the mixture. Degradation of thepolymer may also allow the polymorphic form of CV-8972 to be releasedfrom the mixture. Osmotic pressure due the high concentration gradientof compound between the inside and outside of the mixture may alsocontribute to diffusion of the polymorphic form of CV-8972 from themixture.

For example and without limitation, the polymer may be a cellulosederivative, a gelatin derivative, e.g., a cross-linked gelatinderivative, or a polyester derivative.

Derivatives of cellulose, is a linear chain β(1→4) linked D-glucoseunits, include polymers that contain substitutions on one of more of thehydroxyl groups of each glucose unit. Substituents may be organic orinorganic and are typically attached via ester or ether linkages.Cellulose ester derivatives include carboxymethyl cellulose (CMC), e.g.,sodium carboxymethyl cellulose, ethyl cellulose, ethyl hydroxyethylcellulose, ethyl methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropylmethylcellulose (HPMC), and methylcellulose. Cellulose ether derivativesinclude cellulose acetate, cellulose acetate butyrate, cellulose acetatepropionate, cellulose propionate, cellulose sulfate, cellulosetriacetate, and nitrocellulose. The use of cellulose-based polymers toform biodegradable hydrogels is known in the art and described in, forexample, Sannino, et al., Biodegradable Cellulose-based Hydrogels:Design and Applications, Materials 2009, 2, 353-373;doi:10.3390/ma2020353, the contents of which are incorporated herein byreference.

The mixture may contain multiple polymers or multiple polymeric forms ofthe same polymer. For example, HPMC polymeric forms may differ in avariety of physical properties, including viscosity, degree of methoxylsubstitution, degree of hydroxypropoxyl substitution, or averagemolecule weight.

The viscosity of a HMPC polymeric form may be determined by testingunder standard conditions, including the concentration of HMPC in thesolution and the temperature of the solution. For example and withoutlimitation, the HPMC concentration may be 1%, 1.5%, 2%, 2.5%, or 3%. Forexample and without limitation, the temperature of the solution may be15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C.,24° C., or 25° C.

A polymeric form of a cellulose derivative, such as HPMC, may have adefined viscosity. For example and without limitation, a polymeric formof HPMC may have a viscosity of from about 2 cP to about 4 cP, fromabout 4 cP to about 6 cP, from about 5 cP to about 8 cP, from about 12cP to about 18 cP, from about 40 cP to about 60 cP, from about 80 cP toabout 120 cP, from about 300 cP to about 500 cP, from about 1200 cP toabout 2400 cP, from about 2500 cP to about 5000 cP, from about 9000 cPto about 18,000 cP, from about 12,000 cP to about 24,000 cP, from about12,000 cP to about 24,000 cP, from about 75,000 cP to about 150,000 cP,at least about 2 cP at least about 4 cP at least about 5 cP at leastabout 12 cP at least about 40 cP at least about 80 cP at least about 300cP at least about 1200 cP at least about 2500 cP at least about 9000 cPat least about 12,000 cP at least about 12,000 cP at least about 75,000cP less than about 4 cP, less than about 6 cP, less than about 8 cP,less than about 18 cP, less than about 60 cP, less than about 120 cP,less than about 500 cP, less than about 2400 cP, less than about 5000cP, less than about 18,000 cP, less than about 24,000 cP, less thanabout 24,000 cP, or less than about 150,000 cP for a 2% aqueous solutionof the polymeric form at 20° C.

Polymeric forms of cellulose derivatives, such as HPMC, may vary intheir degree of substitution of the glucose units. The degree ofsubstitution may be expressed as a weight percentage of the substituentor as a molar ratio of substituent to glucose unit. For a cellulosederivative that has two different substituents, such as HPMC, thepolymeric form may be described by the degree of substitution for eachsubstituent.

Each polymeric form of HPMC may independently have a defined degree ofmethoxyl substitution. For example and without limitation, the degree ofmethoxyl substitution may be from about 19% to about 24%, from about 22%to about 24%, from about 27% to about 30%, from about 27% to about 30%,or from about 28% to about 32%.

Each polymeric form of HPMC may independently have a defined degree ofhydroxypropoxyl substitution. For example and without limitation, thedegree of hydroxypropoxyl substitution may be from about 4% to about 8%,from about 7% to about 10%, from about 7% to about 12%, from about 8% toabout 10%, from about 8% to about 11%, or from about 9% to about 12%.

Each polymeric form of HPMC may independently have a defined averagemolecular weight. The average molecular weight may be about 10 kDa,about 13 kDa, about 20 kDa, about 26 kDa, about 41 kDa, about 63 kDa,about 86 kDa, about 110 kDa, about 120 kDa, about 140 kDa, about 180kDa, or about 220 kDa.

When multiple forms of a polymer, such as HPMC, are present, one or morepolymeric forms may be present in a defined amount. For example andwithout limitation, a polymer, such as HPMC, may contain about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about97%, about 98%, about 99%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% by weight of one polymeric form.

Pharmaceutical compositions may include modified-release formulationsthat contain one or more polymorphic forms of CV-8972. The formulationscontain mixtures that include one or more polymorphic forms of CV-8972and one or more erodible polymers that promote swelling of the mixturein an aqueous environment. The hygroscopic and erodible properties ofthe polymers may allow the mixture to form a hydrogel that slowly breaksdown in the digestive tract of the subject. Consequently, the mixturepromotes the steady release of the polymorphic form of CV-8972 andmetabolic products thereof into circulation.

The mixture may contain a defined amount of the polymorphic form ofCV-8972. The mixture may contain at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, or at least 80% by weight of the polymorphic form of CV-8972.

The mixture may contain the polymorphic form of CV-8972 and the polymerin a defined weight ratio. For example and without limitation, themixture may contain the polymorphic form of CV-8972 and the polymer in aweight ratio of about 1:5, about 1:4, about 1:3, about 1:2, about 1:1,about 3:2, about 2:1, about 3:1, about 4:1, about 5:1, from about 1:100to about 100:1, from about 1:100 to about 50:1, from about 1:100 toabout 20:1, from about 1:100 to about 10:1, from about 1:100 to about5:1, from about 1:100 to about 2:1, from about 1:50 to about 100:1, fromabout 1:50 to about 50:1, from about 1:50 to about 20:1, from about 1:50to about 10:1, from about 1:50 to about 5:1, from about 1:50 to about2:1, from about 1:20 to about 100:1, from about 1:20 to about 50:1, fromabout 1:20 to about 20:1, from about 1:20 to about 10:1, from about 1:20to about 5:1, from about 1:20 to about 2:1, from about 1:10 to about100:1, from about 1:10 to about 50:1, from about 1:10 to about 20:1,from about 1:10 to about 10:1, from about 1:10 to about 5:1, from about1:10 to about 2:1, from about 1:5 to about 100:1, from about 1:5 toabout 50:1, from about 1:5 to about 20:1, from about 1:5 to about 10:1,from about 1:5 to about 5:1, from about 1:5 to about 2:1, from about 1:3to about 100:1, from about 1:3 to about 50:1, from about 1:3 to about20:1, from about 1:3 to about 10:1, from about 1:3 to about 5:1, or fromabout 1:3 to about 2:1.

The pharmaceutical composition may be formulated for a particular routeof administration. The pharmaceutical may be formulated for oral,enteral, intravenous, or rectal administration.

The pharmaceutical composition may be formulated as a unit dosagecontaining a defined amount of the polymorphic form of CV-8972. The unitdosage may contain about 5 mg, about 10 mg, about 20 mg, about 50 mg,about 100 mg, about 200 mg, about 500 mg, from about 5 mg to about 10mg, from about 5 mg to about 20 mg, from about 5 mg to about 50 mg, fromabout 5 mg to about 100 mg, from about 5 mg to about 200 mg, from about5 mg to about 500 mg, from about 10 mg to about 20 mg, from about 10 mgto about 50 mg, from about 10 mg to about 100 mg, from about 10 mg toabout 200 mg, from about 10 mg to about 500 mg, from about 20 mg toabout 50 mg, from about 20 mg to about 100 mg, from about 20 mg to about200 mg, from about 20 mg to about 500 mg, from about 50 mg to about 100mg, from about 50 mg to about 200 mg, from about 50 mg to about 500 mg,from about 100 mg to about 200 mg, from about 100 mg to about 500 mg, orfrom about 200 mg to about 500 mg of the polymorphic form of CV-8972.

The pharmaceutical composition may be formulated such that it produces adefined value for one or more parameters, as described below in relationto methods of the invention. For example and without limitation, theparameter may be C_(max), the interval between administration andachieving C_(max), T_(1/2), or AUC.

Pharmaceutical compositions of the invention may contain excipients. Forexample and without limitation, the composition may contain sweeteningagents, flavoring agents, coloring agents, or preserving agents. Thecompositions may contain one or more of mannitol, starch, and magnesiumstearate.

Providing a Polymorph of CV-8972 to a Subject

The invention provides methods of treating a condition in a subject byproviding a polymorph of CV-8972. The polymorph may be Form A, Form B,Form C, Form D, or Form E. The polymorph of CV-8972 may be provided in apharmaceutical composition, as described above. In certain embodimentsof the methods, only a polymorph of Form A is provided.

The polymorph of CV-8972 may be provided by any suitable route or modeof administration. For example and without limitation, the polymorph ofCV-8972 may be provided buccally, dermally, enterally, intraarterially,intramuscularly, intraocularly, intravenously, nasally, orally,parenterally, pulmonarily, rectally, subcutaneously, topically,transdermally, by injection, or with or on an implantable medical device(e.g., stent or drug-eluting stent or balloon equivalents).

The polymorph of CV-8972 may be provided according to a dosing regimen.A dosing regimen may include a dosage, a dosing frequency, or both.

Doses may be provided at any suitable interval. For example and withoutlimitation, doses may be provided once per day, twice per day, threetimes per day, four times per day, five times per day, six times perday, eight times per day, once every 48 hours, once every 36 hours, onceevery 24 hours, once every 12 hours, once every 8 hours, once every 6hours, once every 4 hours, once every 3 hours, once every two days, onceevery three days, once every four days, once every five days, once everyweek, twice per week, three times per week, four times per week, or fivetimes per week.

The dose may contain a defined amount of CV-8972 that improves cardiacmitochondrial function, such as any of the doses described above inrelation to pharmaceutical compositions containing a polymorph ofCV-8972.

The dose may be provided in a single dosage, i.e., the dose may beprovided as a single tablet, capsule, pill, etc. Alternatively, the dosemay be provided in a divided dosage, i.e., the dose may be provided asmultiple tablets, capsules, pills, etc.

The dosing may continue for a defined period. For example and withoutlimitation, doses may be provided for at least one week, at least twoweeks, at least three weeks, at least four weeks, at least six weeks, atleast eight weeks, at least ten weeks, at least twelve weeks or more.

The subject may be a human. The subject may be a human that has acardiovascular condition, rheumatic condition, fibrosis, or cancer. Thesubject may be a human that is at risk of developing a cardiovascularcondition, rheumatic condition, fibrosis, or cancer. A subject may be atrisk of developing a condition if the subject does not meet establishedcriteria for diagnosis of the condition but has one or more symptoms,markers, or other factors that indicate the subject is likely to meetthe diagnostic criteria for the condition in the future. The subject maybe a pediatric, a newborn, a neonate, an infant, a child, an adolescent,a pre-teen, a teenager, an adult, or an elderly subject. The subject maybe in critical care, intensive care, neonatal intensive care, pediatricintensive care, coronary care, cardiothoracic care, surgical intensivecare, medical intensive care, long-term intensive care, an operatingroom, an ambulance, a field hospital, or an out-of-hospital fieldsetting.

Conditions that May be Treated with a Polymorph of CV-8972

The invention provides methods of treating a condition in a subject byproviding a polymorph of CV-8972. The condition may be any disease,disorder, or condition for which increasing mitochondrial energyproduction provides a therapeutic benefit.

The condition may be a cardiac condition. For example and withoutlimitation, the cardiac condition may be aneurysm, angina,atherosclerosis, cardiomyopathy, cerebral vascular disease, congenitalheart disease. coronary artery disease (CAD), coronary heart disease,diabetic cardiomyopathy, heart attack, heart disease, heart failure,high blood pressure (hypertension), ischemic heart disease, pericardialdisease, peripheral arterial disease, refractory angina, rheumatic heartdisease, stable angina, stroke, transient ischemic attack, unstableangina, or valvular heart disease.

Angina pectoris (angina) is chest pain or pressure that is typically dueto insufficient blood flow to the heart muscle. The pain or discomfortis retrosternal or left-sided and may radiate to the left arm, neck,jaw, or back. Several classifications of angina are known.

Stable angina, also called effort angina, is related to myocardialischemia. In stable angina, chest discomfort and associated symptoms areusually triggered by some physical activity, such as running or walking,but symptoms are minimal or non-existent when the patient is at rest orhas taken sublingual nitroglycerin. Symptoms typically abate severalminutes after activity and recur when activity resumes. Symptoms mayalso be induced by cold weather, heavy meals, and emotional stress.

Unstable angina is angina that changes or worsens. Unstable angina hasat least one of the following features: (1) it occurs at rest or withminimal exertion, usually lasting more than 10 minutes, (2) it is severeand of new onset, i.e., within the prior 4-6 weeks, and (3) it occurswith a crescendo pattern, i.e., distinctly more severe, prolonged, orfrequent than before.

Cardiac syndrome X, also called microvascular angina, is angina-likechest pain, in the context of normal epicardial coronary arteries onangiography. Its primary cause is unknown, but factors apparentlyinvolved are endothelial dysfunction and reduced flow in the tinyresistance blood vessels of the heart. Microvascular angina may be partof the pathophysiology of ischemic heart disease.

Refractory angina is a chronic condition (≥3 months in duration) inwhich angina (1) occurs in the context of coronary artery disease (CAD),(2) cannot be controlled by a combination of optimal medical therapy,angioplasty, or bypass surgery, and (3) in which reversible myocardialischemia has been clinically established to be the cause of thesymptoms.

Providing a polymorph of CV-8972 may improve cardiac efficiency in thesubject. A variety of definitions of cardiac efficiency exist in themedical literature. See, e.g., Schipke, J. D. Cardiac efficiency, BasicRes. Cardiol. 89:207-40 (1994); and Gibbs, C. L. and Barclay, C. J.Cardiac efficiency, Cardiovasc. Res. 30:627-634 (1995), incorporatedherein by reference. One definition of cardiac mechanical efficiency isthe ratio of external cardiac power to cardiac energy expenditure by theleft ventricle. See Lopaschuk G. D., et al., Myocardial Fatty AcidMetabolism in Health and Disease, Phys. Rev. 90:207-258 (2010),incorporated herein by reference. Another definition is the ratiobetween stroke work and oxygen consumption, which ranges from 20-25% inthe normal human heart. Visser, F., Measuring cardiac efficiency: is ituseful? Hear Metab. 39:3-4 (2008), incorporated herein by reference.Another definition is the ratio of the stroke volume to mean arterialblood pressure. Any suitable definition of cardiac efficiency may beused to measure the effects of compounds of the invention

A polymorph of CV-8972 may be used to treat a rheumatic disease,disorder, or condition. As used herein, a rheumatic disease, disorder,or condition is any condition that affects the joints, tendons,ligaments, bones, muscles, or connective tissue or is associated withpain in or more of such tissues. The rheumatic disease, disorder, orcondition may primarily affect the joints, tendons, ligaments, bones,muscles, or connective tissue. Examples of such conditions includeankylosing spondylitis, autoimmune-related lung disease, Behcet'sDisease, bursitis, chronic fatigue syndrome, dermatomyositis,fibromyalgia, gout, Gulf War syndrome, infectious arthritis,inflammatory arthritis, inflammatory eye disease, inflammatory myositis,juvenile idiopathic arthritis, lupus, myofascial pain syndrome,osteoarthritis, osteonecrosis of the jaw, osteoporosis, polymyalgiarheumatica, polymyositis, psoriatic arthritis, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, tendinitis, andvasculitis.

The rheumatic disease, disorder, or condition may primarily affect thecardiovascular system and have secondary effects on the joints, tendons,ligaments, bones, muscles, or connective tissue. For example and withoutlimitation, the condition may be alcoholic cardiomyopathy, aneurysm,angina (including refractory angina and angina in the context of heartfailure), atherosclerosis, cardiac fibrosis, cardiomyopathy, cerebralvascular disease, claudication (e.g., peripheral claudication)congenital heart disease. coronary artery disease, coronary heartdisease, cyanotic heart disease, diabetic cardiomyopathy, dilatedcardiomyopathy, heart attack, heart failure, high blood pressure(hypertension), hypertrophic cardiomyopathy, ischemic cardiomyopathy,ischemic heart disease, left ventricular dysfunction, pericardialdisease, peripheral arterial disease, rheumatic heart disease, stroke,transient ischemic attacks, or valvular heart disease.

The rheumatic disease, disorder, or condition may be a rare muscledisease. For example and without limitation, the condition may beCAV3-related distal myopathy, Duchenne Muscular Dystrophy, hypertrophiccardiomyopathy, isolated hyperCKemia, limb-girdle muscular dystrophy 1C,muscle myopathy, myositis, or rippling muscle disease. The rare muscledisease may be associated with a mutation in BICD2, CAV3, or DMD.

The rheumatic disease, disorder, or condition may be a glycogen storagedisease. For example and without limitation, the glycogen storagedisease may be aldolase A deficiency, Andersen disease, Cori's disease,Fanconi-Bickel syndrome, Hers' disease, Lafora disease, McArdle disease,Pompe's disease, Tarui's disease, or von Gierke's disease. The glycogenstorage disease may be associated with a deficiency in an enzyme orprotein, such as acid alpha-glucosidase, aldolase A, β-enolase, glucosetransporter, glucose-6-phosphatase, glycogen branching enzyme, glycogendebranching enzyme, glycogen synthase, glycogenin-1, liver glycogenphosphorylase, muscle glycogen phosphorylase, muscle lactatedehydrogenase, muscle phosphofructokinase, muscle phosphoglyceratemutase, phosphoglycerate mutase, or phosphorylase kinase. The glycogenstorage disease may be associated with a mutation in a gene, such asAGL, ALDOA, ENO3, G6PC, GAA, GBE1, GLUT2, GYG1, GYS2, LDHA, PGAM2,PGAM2, PHKA1, PHKA2, PHKB, PHKG2, PKFM, PYGL, PYGM, or SLC37A4.

The rheumatic disease, disorder, or condition may be another conditionthat affects the joints, tendons, ligaments, bones, muscles, orconnective tissue, such as acute kidney injury, cachexia, chemotherapyinduced nephropathy, contrast nephropathy, disequilibrium, frailty,pulmonary arterial hypertension, pulmonary fibrosis, sarcopenia,tinnitus, or vertigo.

A polymorph of CV-8972 may be used to treat fibrosis or a disease,disorder, or condition associated with fibrosis. In particular, themethods are useful for treating diseases, disorders, or conditions inwhich fibrosis in an organ or tissue is associated with reduced energyproduction by that organ or tissue. The fibrosis may affect any organ ortissue, such as the heart, lungs, liver, brain, cardiovascular system,joints, gastrointestinal system, limbs, digits, skin, bone marrow, orpenis.

The fibrosis may be associated with another condition, e.g., it may besecondary to another condition, or it may lead to the other condition.For example and without limitation, the fibrosis may include or beassociated with adhesive capsulitis, aneurysm, angina, arterialstiffness, arthrofibrosis, atherosclerosis, atrial fibrosis,cardiomyopathy, cerebral vascular disease, cirrhosis, congenital heartdisease. coronary artery disease, coronary heart disease, Crohn'sdisease, cystic fibrosis, diabetic cardiomyopathy, Dupuytren'scontracture, endomyocardial fibrosis, glial scar, heart attack, heartfailure, high blood pressure (hypertension), idiopathic pulmonaryfibrosis, ischemic heart disease, keloid, mediastinal fibrosis,myelofibrosis, nephrogenic systemic fibrosis, old myocardial infarction,pericardial disease, peripheral arterial disease, Peyronie's disease,progressive massive fibrosis, pulmonary fibrosis, radiation-induced lunginjury, retroperitoneal fibrosis, rheumatic heart disease, scleroderma,stroke, systemic sclerosis transient ischemic attacks, or valvular heartdisease.

A polymorph of CV-8972 may be used to treat cancer. For example andwithout limitation, the cancer may be bladder cancer, brain cancer,breast cancer, carcinoma, cervical cancer, colon cancer, colorectalcancer, gastric cancer, glioblastoma, glioma, head and neck cancer,kidney cancer, leukemia, liposarcoma, liver cancer, lung cancer,lymphoma, medullablastoma, melanoma, muscle cancer, neuroblastoma,oligoastrocytoma, oligodendroglioma, osteosarcoma, ovarian cancer,pancreatic cancer, paraganglioma, prostate cancer, sarcoma, or thyroidcancer.

EXAMPLES Example 1

Summary

A comprehensive polymorph screening for CV-8972, which has the structureof Formula (X), was undertaken. The CV-8972 starting material wascharacterized by X-ray powder diffraction (XRPD), thermogravimetricanalysis (TGA), differential scanning calorimetry (DSC), dynamic vaporsorption (DVS), and polarized light microscopy (PLM). The data showedthat the material is crystalline in nature and has similar XRPD patternto that of the Form A. Starting with Form A, polymorph/single crystalscreening experiments were set up under 34 conditions using methods ofvapor diffusion, slow evaporation, and cooling crystallization. Fiveunique XRPD patterns were observed, which include Form A, Form B, FormsA+C, Form D, and Form E. Form A is monohydrate form as confirmed bysingle crystal structure. Form D is anhydrous, and it was also confirmedby single crystal structure. Form E is an anhydrous form producedthrough dehydration of Form A at ˜90° C. Form B is a known anhydratefrom a separate study. Form C was not obtained in the pure form duringthe study but rather appeared as a mixture of Forms A+C. Water activityanalysis indicated that Form E converts to Form A under all conditionstested. In addition, when Form E was exposed to ambient temperature andhumidity, it showed partial conversion to Form A. Further, the resultsfrom slurry competition between both anhydrous Forms D and E alsoindicated that both forms converted to Form A during the experiments.These results suggest that the Form A is the most stable form at theambient temperature and humidity.

Characterization of Form A

The starting material of CV-8972 was characterized using X-ray powderdiffraction (XRPD), thermogravimetric analysis (TGA), differentialscanning calorimetry (DSC), and polarized light microscopy (PLM).

FIG. 4 is an XRPD diffractogram of the CV-8972 starting material. TheXRPD results suggested high crystallinity of the starting material.Comparison of the XRPD of the starting material with previously knownpolymorphs indicated that it is Form A.

FIG. 5 shows TGA and DSC thermograms of the CV-8972 starting material.TGA thermogram is shown in green, and DSC thermogram is shown in blue.As shown by the TGA and DSC data, about 3.46% weight loss was observedup to 150° C. before decomposition. DSC showed a small endotherm at85.3° C. (peak) and a possible melting endotherm at 214.6° C. (onset),followed by decomposition. and a melting point at 131.7° C. (peak) wasobserved.

FIG. 6 shows XRPD diffractograms of various forms of CV-8972. CV-8972starting material is shown in blue; CV-8972 following incubation at 90°C. for 8 hours is shown in red; and CV-8972 following incubation at 65°C. in a vacuum for 2 hours is shown in purple. To see if the small DSCendotherm at 85.3° C. corresponds to polymorphic phase transition ordehydration, XRPD was performed on Form A after storing it in an oven at90° C. for 8 hours. The data showed that the Form A converts to Form E.

FIG. 7 is a polarized microscopic image of CV-8972 starting material.Very platy “mica-like” morphology of the crystals was observed by PLM.

FIG. 8 is a dynamic vapor sorption isotherm plot. Cycle sorption isshown in red; cycle 1 desorption is shown in blue; and cycle 2 sorptionis shown in green. DVS results showed that water uptake of CV-8972 is<0.2% at 25° C. and 80% relative humidity (RH) indicated that startingmaterial was non hygroscopic. However, there is a drastic increase inthe mass change beyond 80% RH, which indicates there could bedeliquescence.

FIG. 9 shows XRPD diffractograms of CV-8972 before and after dynamicvapor sorption. Pre-DVS data is shown in red; and post-DVS data is shownin blue. The XRPD of the sample after DVS indicated weak crystallinepeaks but was mostly similar to the starting material.

FIG. 10 shows XRPD diffractograms of CV-8972 in its dehydrated andrehydrated forms. Data from starting material is shown in blue; datafollowing incubation for 2 hours in vacuum oven are shown in red; anddata from heated material that was exposed to ambient relative humidityis shown in green. To monitor Form A in its dehydrated state, it wasplaced in a vacuum oven at 65° C. for 2 hours followed by its XRPDanalysis. The XRPD results showed that this process created a newanhydrous form of the material and assigned as Form E. Rehydration ofForm E when exposed to ambient RH resulted in its partial conversion toForm A.

X-ray powder diffraction data has been challenging to interpret due tothe extreme preferred orientation, which results in large variations inthe peak intensities from one sample preparation to the next. Tominimize this effect, single crystal X-ray diffraction was used toacquire the crystallographic structure, and the X-ray powder diffractionthat should be observed in an ideal sample that is absent of preferredorientation was calculated.

Polymorph/Single Crystal Screening

Starting with Form A, polymorph screening experiments were set up under34 conditions using methods of slurry conversion, liquid vapordiffusion, slow evaporation, and slow cooling. The approximatesolubility of starting material was determined at room temperature (RT).Accurately weighed samples of approximately 2 mg weight were added intoa 3-mL glass vial. Solvents were then added step wise (50/50/200/700 μL)into the vials until the solids were dissolved or a total volume of 1 mLwas reached. The solubility of the starting material in various solventsis shown in Table 1.

TABLE 1 Approximate solubility of starting material (6010242-01-A) at RTExperiment ID Solvent (v:v) Solubility (mg/mL) 6010242-02-A1 n-Heptane S< 1.9 6010242-02-A2 ACN S < 1.5 6010242-02-A3 MIBK S < 2.0 6010242-02-A4EtOAc S < 1.8 6010242-02-A5 THF S < 2.2 6010242-02-A6 EtOH S < 1.76010242-02-A7 Acetone S < 2.0 6010242-02-A8 MEK S < 2.0 6010242-02-A9IPA S < 2.3 6010242-02-A10 CHCI₃ S < 1.8 6010242-02-A11 IPAc S < 1.76010242-02-A12 1,4-Dioxane S < 1.7 6010242-02-A13 CPME S < 1.56010242-02-A14 DCM S < 2.2 6010242-02-A15 Toluene S < 2.3 6010242-02-A16DMSO 5.0 < S < 15 6010242-02-A17 DMF 1.8 < S < 6.0 6010242-02-A18 NMP6.7 < S < 20 6010242-02-A19 H₂O S > 44.0 6010242-02-A20 MeOH S > 46.0

Results from solubility analysis were used to guide the solventselection in polymorph screening. Polymorph screening experiments wereperformed using different crystallization or solid transition methods.Polymorph screening experiments are summarized in Table 2.

TABLE 2 Method No. of Experiments Crystal Type Liquid vapor diffusion 24Form A, B, C, D, and E Slow evaporation 2 Gel Slow cooling 2 Noprecipitation Slurry conversion 6 Form A Total 34 Form A, B, C, D, and E

FIG. 11 shows XRPD diffractograms of various polymorphs of CV-8972. FormA is shown in blue; Form B is shown in green; a mixture of Forms A and Cis shown in navy; Form D is shown in orange; and Form E is shown inpurple.

The various crystal forms of CV-8972 are summarized in Table 3.

TABLE 3 Form Crystal Form Method of obtaining Identification Form AStarting material Monohydrate* Form B Liquid vapor diffusion of MTBEHydrate of unknown in MeOH stoichiometry^(#) Form C Multiple methods aslisted below Solvate/hydrate (mixed with Form A) Form D Liquid vapordiffusion of IPA in Anhydrate* MeOH Form E 65° C. Vacuum for 2 hoursAnhydrate *Single crystal structures for these forms are available andare provided as separate reports ^(#)information obtained from separatereport

Liquid vapor diffusion experiments were conducted in different solventconditions. Approximately 15-25 mg of starting material was dissolved inan appropriate solvent to obtain a clear solution in a 3-mL vial. Thissolution was then placed into a 20-mL vial with 3 mL of volatilesolvents. The 20-mL vial was sealed with a cap and kept at RT allowingenough time for organic vapor to interact with the solution. Theprecipitates were isolated for XRPD analysis. Results from liquid vapordiffusion experiments are summarized in Table 4.

TABLE 4 Anti- Experiment ID Solvent solvent Observation 6010242-03-A1H₂O MEK Amorphous 6010242-03-A2 1,4-Dioxane Gel 6010242-03-A3 THF FormA + C 6010242-03-A4 Acetone Form A + C 6010242-03-A5 ACN Form A + C6010242-03-A6 EtOH Form A + C 6010242-03-A7 IPA Clear 6010242-03-A8 MeOHMEK Form A + C 6010242-03-A9 MTBE Single crystal (Form B) 6010242-03-A10THF Gel 6010242-03-A11 Acetone Gel 6010242-03-A12 ACN Gel 6010242-03-A13EtOH Form A + C 6010242-03-A14 IPA Single crystal (Form D)6010242-03-A15 EtOAc Amorphous solid 6010242-07-A1 MeOH IPA Form E6010242-07-A2 IPA Form D 6010242-07-A3 IPA Form D 6010242-07-A4 TolueneForm A + C 6010242-07-A5 Dioxane Small single crystal on the wall (FormA + C) 6010242-07-A6 MIBK Small single crystal on the wall (Form A + C)6010242-04-A1 H₂O MTBE Gel 6010242-04-A2 H₂O IPA Gel 6010242-04-A3 MeOHDCM Small single crystal on the wall (Form A + C)

Slow evaporation experiments were performed under various conditions.Briefly, a saturated solution of starting material prepared in differentsolvents was added to a HPLC vial. The visually clear solutions werecovered by Parafilm® with 5-10 pinholes and subjected to evaporation atRT. The solids were isolated for XRPD analysis. Results from slowevaporation experiments are summarized in Table 5.

TABLE 5 Experiment ID Solvent (v:v) Solid Form 6010242-06-A1 MeOH Whitesolid poor crystalline 6010242-06-A2 H₂O Gel

Slow cooling experiments were conducted in two different solventsystems. About 10˜15 mg of starting material was suspended inappropriate solvent in a 2-mL glass vial at RT. The suspension was thenheated to 50° C., equilibrated for about two hours and filtered using anylon membrane (pore size of 0.22 μm). Each filtrate was slowly cooleddown to 5° C. at a rate of 0.1° C./min. Results from slow coolingexperiments are summarized in Table 6.

TABLE 6 Experiment ID Solvent (v:v) Solid Form 6010242-05-A1 DMSO Noprecipitation 6010242-05-A2 NMP No precipitation

Slurry conversion experiments were conducted at RT in different solventsystems. Approximately 20 mg of starting material was suspended in 0.1mL of solvent in HPLC vials. After the suspension was stirredmagnetically for 48 hours at RT, the remaining solids were isolated forXRPD analysis. Results from slurry conversion experiments are summarizedin Table 7.

TABLE 7 Experiment ID Solvent (v:v) Solid Form 6010242-15-A1 AcetoneForm A 6010242-15-A2 Acetone/H₂O (a_(w) = 0.2, 941/59) Form A6010242-15-A3 Acetone/H₂O (a_(w) = 0.4, 857/143) Form A 6010242-15-A4Acetone/H₂O (a_(w) = 0.6, 726/274) Form A 6010242-15-A5 Acetone/H₂O(a_(w) = 0.8, 492/508) Form A 6010242-15-A6 H₂O Form A 6010242-15-A7Form D + E in acetone Form A

Conclusions

The Form A was successfully characterized to understand its formbehavior. A comprehensive polymorph screening in 34 different conditionswas performed. Five polymorph of the CV-8972 were identified during thescreening, including Form A, Form B, a mixture of Forms A+C, Form D, andForm E. Form D and E are anhydrous, Form A is a monohydrate, and Form Bis a hydrate with unknown stoichiometry. Phase origin of Form C is notknown since it was not obtained it in pure form; it always crystallizedas mixture with Form A. Based on polymorph screening it is apparent thatCV-8972 has a tendency to form multiple polymorphs. Current studies haveconcluded that Form A is the best form for development of CV-8972 and isa stable monohydrate form, and is the most stable form under conditionsof ambient temperature and humidity.

Instruments and Methods

Starting material was used to analyze Form A and screen for otherpolymorphs.

XRPD was performed with a Panalytical X'Pert3 Powder XRPD on a Sizero-background holder. The 2θ position was calibrated against aPanalytical Si reference standard disc. Instrumental parameters used forXPRD are listed in Table 8.

TABLE 8 Parameters Reflection Mode X-Ray wavelength Cu, kα Kα1 (Å):1.540598, Kα2 (Å): 1.544426, Kα2/Kα1 intensity ratio: 0.50 X-Ray tubesetting 45 kV, 40 mA Divergence slit Fixed 1/8° Scan mode ContinuousScan range 3-40 (°2TH) Scan step time [s] 18.87 Step size (°2TH) 0.0131Test Time 4 min 15 s

TGA data was collected using a TA Discovery 550 TGA from TA Instrument.DSC was performed using a TA Q2000 DSC from TA Instrument. DSC wascalibrated with Indium reference standard and the TGA was calibratedusing nickel reference standard. Detailed parameters used for TGA andDSC are listed in Table 9.

TABLE 9 Parameters TGA DSC Method Ramp Ramp Sample pan Platinum, openAluminum, crimped Temperature RT - 300° C. Heating rate 10° C./min Purgegas N₂

Polarized light microscopic (PLM) pictures were captured on a NikonDS-Fi2 upright microscope at room temperature. Low viscosity microscopeimmersion oil (Resolve®) was used to disperse powder crystals.

Example 2

Summary

To determine the crystal structure of CV-8972, a single monohydratecrystal was grown, and a suitable single crystal was used for a fullcrystal X-ray diffraction (SCXRD) data collection at 199 K. A crystalstructure with a R₁ value of 0.0303 (I>2σ(I)) was obtained. Thestructure showed that this crystal form is a monohydrate, tri-HCl salt.

Crystal Growth and SCXRD Preparation

Single crystals of C₂₂H₃₄Cl₃N₃O₆ (CV-8972) were obtained via slowcooling: 163.2 mg starting material was weighed into a 2-mL glass vial,and 0.100 mL water was added to dissolve the solids at 50° C., then thesolution was slowly cooled to 10° C. over 12 hours before harvesting.

FIG. 12 is a PLM image of a batch of single crystals of C₂₂H₃₄Cl₃N₃O₆(CV-8972). Bar represents 100 μm.

FIG. 13 shows PLM images of a crystal used for single-crystaldiffractometer. Bars represent 100 μm. A thick needle was picked out andtrimmed down to a size of 200×160×100 μm uniform block. This sample wasmounted on a 100 mm MiTeGen MicroLoop™ with low viscosity cryo-oil(MiTeGen LV CryoOil™).

FIG. 14 shows images of a crystal mounted on a 100 micro Mitegen loop onthe diffractometer.

Single Crystal Structure Determination

A total of 9576 frames were collected using Bruker Apex3 v2018-7.2. Thetotal exposure time was 18 hours (exposure times were adjusted based on2θ). The frames were integrated with the Bruker SAINT software packageusing a narrow-frame algorithm. The integration of the data using anorthorhombic unit cell yielded a total of 157237 reflections to amaximum θ angle of 81.01° (0.78 Å resolution), of which 5641 wereindependent (average redundancy 27.874, completeness=99.8%,R_(int)=4.41%, R_(sig)=1.34%) and 5388 (95.51%) were greater than2σ(F²). The final cell constants of a=7.8826(2) Å, b=12.4776(3) Å,c=52.3580(13) Å, volume=5149.7(2) Å³, are based upon the refinement ofthe XYZ-centroids of 1406 reflections above 20 σ(I) with11.75°<2θ<100.6°. Data were corrected for absorption effects using theMulti-Scan method (SADABS). The ratio of minimum to maximum apparenttransmission was 0.788. The calculated minimum and maximum transmissioncoefficients (based on crystal size) are 0.5340 and 0.7160.

The structure was solved and refined using the Olex2 incorporatingSHELXTL Software Package using the orthorhombic space group Pbca, withZ=8 for the formula unit, C₂₂H₃₄Cl₃N₃O₆. One asymmetric unit containsone whole API molecule. The final anisotropic full-matrix least-squaresrefinement on F² with 330 variables (0 restraints) converged atR₁=3.03%, for the observed data and wR2=7.98% for all data. Thegoodness-of-fit was 1.041. The largest peak in the final differenceelectron density synthesis was 0.358 e−/Å³ (0.81 Å from Cl₁) and thelargest hole was −0.438 e−/Å³ (0.66 Å from Cl₁). Most of the positionsand thermal ellipsoids of hydrogens were treated as riding models (AFIX23, AFIX 43, and AFIX 137 used). However, the crucial hydrogen atomsinvolving hydrogen bonding and salt formation were refined freelywithout any constraints. On the basis of the final model, the calculateddensity was 1.400 g/cm³ and F(000), 2288 e−. Crystallographic parametersof the C₂₂H₃₄Cl₃N₃O₆ crystal are summarized in Table 10.

TABLE 10 Identification code 6010242_13 Chemical formula C₂₂H₃₄Cl₃N₃O₆Formula weight 542.87 g/mol Wavelength 1.54178 Å Temperature 199.0 KCrystal size 0.10 × 0.16 × 0.20 mm Crystal habit colorless trimmedneedle Crystal system orthorhombic Space group Pbca Unit cell dimensionsa = 7.8826(2) Å α = 90° b = 12.4776(3) Å β = 90° c = 52.3580(13) Å γ =90° Volume 5149.7(2) Å³ Z 8 Density (calculated) 1.400 g/cm³ Absorption3.583 mm⁻¹ coefficient(μ(CuKα)) F(000) 2288 Theta range for datacollection 3.38 to 81.01° Index ranges −10 <= h <= 10, −15 <= k <= 15,−66 <= 1 <= 66 Reflections collected 157237 Independent reflections 5641[R(int) = 0.0441] Coverage of independent 99.8% reflections Absorptioncorrection Multi-Scan Max. and min. transmission 0.7160 and 0.5340Structure solution technique direct methods Structure solution programXS (Sheldrick, 2008) Refinement method Full-matrix least-squares on F²Refinement program XL (Sheldrick, 2008) Function minimized Σ w(F_(o) ² −F_(c) ²)² Data/restraints/parameters 5641/0/330 Goodness-of-fit on F²1.041 Δ/σ_(max) 0.001 Final R indices 5388 data; I > 2σ(I) R₁ = 0.0303,wR2 = 0.0783 all data R₁ = 0.0318, wR2 = 0.0798 Weighting scheme w =1/[σ²(F_(o) ²) + (0.0364 P)² + 2.7627 P] where P = (F_(o) ² + 2F_(c)²)/3 Largest diff. peak and hole 0.358 and −0.438 eÅ⁻³ R.M.S. deviationfrom mean 0.045 eÅ⁻³

FIG. 15 is an Ortep diagram of an asymmetric unit of the C₂₂H₃₄Cl₃N₃O₆crystal. The Ortep diagram of an asymmetric unit of the C₂₂H₃₄Cl₃N₃O₆crystal demonstrates that this API is a monohydrate tris-HCl salt, as aratio of 1:3:1 (API:HCl:H₂O) was observed.

FIG. 16 shows one unit cell of the C₂₂H₃₄Cl₃N₃O₆ crystal.

FIG. 17 is a diagram of hydrogen bonds networks and counter-ion pairs inthe C₂₂H₃₄Cl₃N₃O₆ crystal. The diagram shows that the threehydrochloride molecules are deprotonated, whereas the three nitrogensare protonated. The water molecule serves as a hydrogen bond donor tobridging two chlorine anions. The crystallographic measurements of thehydrogen bonds and counter-ion pairs in the C₂₂H₃₄Cl₃N₃O₆ crystal aresummarized in Table 11.

TABLE 11 D-H . . . A d(D . . . A)/Å (D-H . . . A)/° d(D . . . A) <(DHA)O6—H6A . . . Cl3^(#1) 0.85(3) 2.37(3) 3.1997(18) 167.(2) O6—H6B . . .Cl2^(#2) 0.91(3) 2.27(3) 3.1815(16) 176.(2) N2—H2 . . . Cl1^(#3)0.932(18) 2.119(18) 3.0499(11) 177.1(16) N1—H1 . . . Cl2^(#4) 0.919(17)2.127(18) 3.0462(11) 179.2(16) N3—H3 . . . Cl3 0.95(2) 2.07(2)2.9891(13) 163.9(19) Symmetry transformations used to generateequivalent atoms: ^(#1)2 − X, 0.5 + Y, 0.5 − Z; ^(#2)0.5 + X, +Y, 0.5 −Z; ^(#3)1 + X, +Y, +Z; $4: 1.5 − X, 0.5 + Y, +Z

The final cif file was checked with Platon using Olex2 locally, only onelevel C alert was found (missing three reflections), together with eightlevel G alerts. An extensive data collection strategy was implemented toprevent this issue, and the completeness and redundancy of this datasetwere 99.8% and 27.87, respectively.

FIG. 18 shows calculated and measured XRPD diagrams of the C₂₂H₃₄Cl₃N₃O₆crystal. Calculated XRPD diffractogram is shown in red; and measuredXRPD diffractogram is shown in blue. Powder x-ray diffraction of thisbatch was obtained and compared with a calculated pattern based on thiscrystal structure using Mercury. The experimental peak positions andintensities fit well with the calculated pattern.

Instruments and Methods

The X-ray intensity data were measured at 199.0 K (controlled by OxfordCryostream 800) on a Bruker Venture X-ray diffractometer. IncoatecMicrofocus Source (IμS 3.0) monochromated Cu Kα radiation (λ=1.54178 Å,voltage=50 kV, current=1.1 mA) was used as the x-ray source. Theintensity data was collected by a Photon II detector.

Polarized light microscopic picture was captured on Nikon DS-Fi2 uprightmicroscope at room temperature.

XRPD was performed with a Panalytical X'Pert3 Powder XRPD on a Sizero-background holder. The 2θ position was calibrated against aPanalytical Si reference standard disc.

Example 3

Summary

To determine the crystal structure of CV-8972, a single anhydratecrystal of CV-8972 was grown, and a suitable single crystal was used fora full SCXRD data collection at 102 K. A crystal structure with a R₁value of 0.0328 (I>2σ(I)) was obtained. The structure showed that thiscrystal form is an anhydrous tri-HCl salt.

Crystal Growth and SCXRD Preparation

Single crystals of anhydrous tri-HCl salt of CV-8972 were obtained vialiquid vapor diffusion of MTBE in MeOH solution. Briefly, a saturatedsolution of CV-8972 in MeOH was obtained at RT and filled into 2-mLglass vial, which was then kept inside a bigger 20 mL vial having 2 mLof MTBE. The vial was taken out when it showed presence of whitecrystalline material.

FIG. 19 shows PLM images of single anhydrous crystals fromrecrystallized CV-8972.

FIG. 20 is an image of a single anhydrous crystal from recrystallizedCV-8972 mounted on a tip of a glass fiber. The colorless crystal wassubsequently set up on the SCXRD instrument.

Single Crystal Structure Determination

A colorless crystal was mounted on a tip of a glass fiber. The X-rayintensity data were measured at 102K temperature on a Bruker D8 QuestPHOTON 100 CMOS X-ray diffractometer system with Incoatec MicrofocusSource (IμS) monochromated Mo Kα radiation (λ=0.71073 Å, sealed tube)using omega/phi-scan technique. The data were collected in 1660 frameswith 10 second exposure times. Crystallographic data: C₂₂H₃₂O₅N₃Cl₃:a=6.9940(6) Å, b=10.5742(9) Å, c=17.5786(14) Å, α=78.252(2)°,β=82.823(2)°, y=82.476(2)°, V=1255.37(18) Å³, Z=2, F.W.=524.85, μ=0.403mm−1, d=1.389 g/cm³, F(000)=552.

Crystal data and structure refinement for j1_a are provided in Table 12.

TABLE 12 j1_a Crystal data Chemical formula C₂₂H₃₂Cl₃N₃O₅ M_(r) 524.85Crystal system, space group Triclinic, P⁻1 Temperature (K) 102 a, b, c(Å) 6.9940 (6), 10.5742 (9), 17.5786 (14) α, β, γ (°) 78.252 (2), 82.823(2), 82.476 (2) V (Å³) 1255.37 (18) Z 2 Radiation type Mo Kα μ (mm⁻¹)0.40 Crystal size (mm) 0.29 × 0.23 × 0.06 Data collection DiffractometerBruker D8 Quest PHOTON 100 CMOS Absorption correction Multi-scan BRUKERSADABS T_(min), T_(max) 0.687, 0.747 No. of measured, independent 30843,8732, 7553 and observed [I > 2σ(I)] reflections R_(int) 0.027 (sinθ/λ)_(max) (Å⁻¹) 0.746 Refinement R[F² > 2σ(F²)], wR(F²), S 0.033,0.098, 1.01 No. of reflections 8732 No. of parameters 310 No. ofrestraints 3 H-atom treatment H atoms treated by a mixture ofindependent and constrained refinement Δ 

 max, Δ 

 min (e Å⁻³) 0.55, −0.46

Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å²×10³) for j1_a are provided in Table 13. U(eq) is definedas one third of the trace of the orthogonalized Uij tensor

TABLE 13 x y z U(eq) Cl(1) 3641(1) 9477(1) 6048(1) 14(1) Cl(2) 11992(1)5170(1) 5985(1) 13(1) Cl(3) 1821(1) 10077(1) 2138(1) 17(1) O(1) 11846(1)6620(1) 2868(1) 16(1) O(2) 9860(1) 7793(1) 3643(1) 12(1) O(3) 7371(1)5399(1) 8524(1) 14(1) O(4) 6912(1) 6708(1) 9782(1) 14(1) O(5) 4066(1)8571(1) 9907(1) 16(1) N(1) 5606(1) 8451(1) 2202(1) 15(1) N(2) 9414(1)7581(1) 5446(1) 9(1) N(3) 6144(1) 6997(1) 6601(1) 9(1) C(1) 5518(2)7647(1) 1711(1) 18(1) C(2) 6990(2) 6666(1) 1628(1) 18(1) C(3) 8550(2)6526(1) 2067(1) 15(1) C(4) 8618(2) 7383(1) 2564(1) 12(1) C(5) 7105(2)8356(1) 2629(1) 14(1) C(6) 10306(2) 7218(1) 3030(1) 12(1) C(7) 11386(2)7722(1) 4138(1) 13(1) C(8) 10548(2) 8407(1) 4797(1) 11(1) C(9) 8935(2)8244(1) 6132(1) 11(1) C(10) 7968(2) 7358(1) 6820(1) 11(1) C(11) 6595(2)6347(1) 5908(1) 11(1) C(12) 7583(1) 7222(1) 5220(1) 10(1) C(13) 5051(2)6149(1) 7265(1) 12(1) C(14) 4672(2) 6764(1) 7977(1) 11(1) C(15) 5894(1)6385(1) 8580(1) 11(1) C(16) 5670(1) 7028(1) 9206(1) 11(1) C(17) 4153(2)8022(1) 9262(1) 12(1) C(18) 2891(2) 8375(1) 8683(1) 13(1) C(19) 3177(2)7754(1) 8045(1) 12(1) C(20) 7254(2) 4307(1) 9160(1) 17(1) C(21) 8819(2)7087(1) 9516(1) 18(1) C(22) 2481(2) 9539(1) 10007(1) 21(1)

Bond lengths [Å] for j1_a are provided in Table 14.

TABLE 14 O(1)—C(6) 1.2075(13) O(2)—C(6) 1.3282(12) O(2)—C(7) 1.4443(13)O(3)—C(15) 1.3774(12) O(3)—C(20) 1.4371(14) O(4)—C(16) 1.3774(13)O(4)—C(21) 1.4372(14) O(5)—C(17) 1.3658(13) O(5)—C(22) 1.4268(14)N(1)—H(1) 0.868(14) N(1)—C(1) 1.3404(15) N(1)—C(5) 1.3460(14) N(2)—H(2)0.881(13) N(2)—C(8) 1.4972(13) N(2)—C(9) 1.4964(13) N(2)—C(12)1.5025(13) N(3)—H(3) 0.868(13) N(3)—C(10) 1.4937(13) N(3)—C(11)1.4993(13) N(3)—C(13) 1.5099(13) C(1)—C(2) 1.3793(17) C(2)—C(3)1.3893(16) C(3)—C(4) 1.3898(15) C(4)—C(5) 1.3874(15) C(4)—C(6)1.4909(15) C(7)—C(8) 1.5071(14) C(9)—C(10) 1.5162(14) C(11)—C(12)1.5160(14) C(13)—C(14) 1.5052(14) C(14)—C(19) 1.3915(14) C(14)—C(15)1.4062(14) C(15)—C(16) 1.3903(14) C(16)—C(17) 1.4014(14) C(17)—C(18)1.3922(15) C(18)—C(19) 1.3926(15)

Angles [deg] for j1_a are provided in Table 15.

TABLE 15 C(6)—O(2)—C(7) 116.27(8) C(15)—O(3)—C(20) 113.71(8)C(16)—O(4)—C(21) 112.87(8) C(17)—O(5)—C(22) 116.45(9) H(1)—N(1)—C(1)115.0(12) H(1)—N(1)—C(5) 122.2(12) C(1)—N(1)—C(5) 122.73(10)H(2)—N(2)—C(8) 108.6(11) H(2)—N(2)—C(9) 105.1(11) C(8)—N(2)—C(9)110.24(8) H(2)—N(2)—C(12) 109.0(11) C(8)—N(2)—C(12) 114.11(8)C(9)—N(2)—C(12) 109.30(8) H(3)—N(3)—C(10) 107.1(11) H(3)—N(3)—C(11)107.3(11) C(10)—N(3)—C(11) 109.71(8) H(3)—N(3)—C(13) 108.8(11)C(10)—N(3)—C(13) 113.17(8) C(11)—N(3)—C(13) 110.51(8) N(1)—C(1)—C(2)120.40(11) C(1)—C(2)—C(3) 118.57(10) C(2)—C(3)—C(4) 119.88(10)C(5)—C(4)—C(3) 119.57(10) C(5)—C(4)—C(6) 121.37(9) C(3)—C(4)—C(6)119.05(9) N(1)—C(5)—C(4) 118.85(10) O(1)—C(6)—O(2) 125.57(10)O(1)—C(6)—C(4) 123.74(10) O(2)—C(6)—C(4) 110.67(9) O(2)—C(7)—C(8)107.00(8) N(2)—C(8)—C(7) 113.78(8) N(2)—C(9)—C(10) 110.57(8)N(3)—C(10)—C(9) 109.72(8) N(3)—C(11)—C(12) 111.10(8) N(2)—C(12)—C(11)109.69(8) C(14)—C(13)—N(3) 110.97(8) C(19)—C(14)—C(15) 118.22(9)C(19)—C(14)—C(13) 121.58(9) C(15)—C(14)—C(13) 120.14(9) O(3)—C(15)—C(16)120.40(9) O(3)—C(15)—C(14) 118.90(9) C(16)—C(15)—C(14) 120.65(9)O(4)—C(16)—C(15) 121.32(9) O(4)—C(16)—C(17) 118.62(9) C(15)—C(16)—C(17)120.07(9) O(5)—C(17)—C(18) 125.51(9) O(5)—C(17)—C(16) 114.73(9)C(18)—C(17)—C(16) 119.75(9) C(17)—C(18)—C(19) 119.51(10)C(18)—C(19)—C(14) 121.71(10)

Of the 8732 unique reflections collected to a maximum theta angle of32.02° (0.67 Å resolution), 7553 were observed (I>2σ(I)). The linearabsorption coefficient for Mo Kα radiation is 0.403 mm⁻¹. The data wereintegrated with the manufacturer's SAINT software and corrected forabsorption effects using the Multi-Scan method (SADABS).

Subsequent solution and refinement were performed using the SHELXTL-2014solution package operating on a Pentium computer. The structure wassolved by direct method using SHELXTL-2014 Software Package.Non-hydrogen atomic scattering factors were taken from the literaturetabulations. Non-hydrogen atoms were located from successive differenceFourier map calculations. In the final cycles of each refinement, allthe non-hydrogen atoms were refined in anisotropic displacementparameters. Except for H(1), H(2), H(3) on N(1), N(2), N(3) atoms of themolecule that were located from difference Fourier map and refined withproper restraints, the rest of hydrogen atom positions were calculatedand allowed to ride on the carbon to which they are bonded, assuming aC—H bond length of m Å (m=0.990 for CH₂ groups, m=0.980 for CH₃ groups,m=0.950 for Ph-H groups). Hydrogen atom temperature factors were fixedat n (n=1.2 for CH₂, Ph-H groups, n=1.5 for CH₃) times the isotropictemperature factor of the C-atom to which they are bonded. The crystalsystem of compound is triclinic, space group P-1 (No. 2) and the finalresidual values based on 310 variable parameters and 7553 observedreflections (I>2σ(I)) are R₁=0.0328, wR2=0.0926, and those for allunique reflections are R₁=0.0408, wR2=0.0975. The goodness-of-fitindicator for all data is 1.014. Peaks on the final difference map,ranging from 0.549 to −0.459 e/Å³, are of no chemical significance. Theefforts have been made to resolve as many alerts as possible generatedby CheckCIF program. The current highest alerts are at level G.

FIG. 21 is a thermal ellipsoid diagram of an asymmetric unit of theC₂₂H₃₂C₁₃N₃O₅ crystal. The diagram demonstrates that this form is ananhydrate, tris-HCl salt form.

FIG. 22 shows one unit cell of the C₂₂H₃₂C₁₃N₃O₅ crystal.

FIG. 23 is a diagram of hydrogen bonds networks and counter-ion pairs inthe C₂₂H₃₂C₁₃N₃O₅ crystal.

FIG. 24 shows calculated and measured XRPD diagrams of the C₂₂H₃₄Cl₃N₃O₆crystal. Calculated XRPD diffractogram is shown in red; and measuredXRPD diffractogram is shown in green.

The compound crystallizes in triclinic, space group P-1 (No. 2). Theasymmetric unit contains one molecule in the form of cation/anion salt(an an hydrate tri-HCl salt) with formula of C₂₂H₃₂O₅N₃Cl₃. There mightbe some intra-molecular H-bonding between N(1)-H(1) . . . C₁(3) (withdistance of 2.9634(10)), N(2)-H(2) . . . C₁(2) (with distance of2.9822(9)), N(3)-H(3) . . . C₁(1) (with distance of 3.0120(9)).Structure solution, refinement and the calculation of derived resultswere performed using the SHELXTL-2014 package of computer programs.Neutral atom scattering factors were those of Cromer and Waber, and thereal and imaginary anomalous dispersion corrections were those ofCromer.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification, and guidance that can be adapted to the practice ofthis invention in its various embodiments and equivalents thereof.

1.-20. (canceled)
 21. A pharmaceutical composition comprising a Form Apolymorph of a compound of Formula (X):

wherein the Form A polymorph has an endothermic peak at about 85.3° C.(±5° C.) and at about 214.6° C. (±5° C.) in a differential scanningcalorimetry (DSC) thermogram.
 22. The pharmaceutical composition ofclaim 21, wherein the pharmaceutical composition comprises ahydrochloride salt of the compound.
 23. The pharmaceutical compositionof claim 22, wherein the pharmaceutical composition comprises a hydratedform of the compound.
 24. The pharmaceutical composition of claim 23,wherein the hydrated form of the compound is a monohydrate.
 25. Thepharmaceutical composition of claim 22, wherein the pharmaceuticalcomposition comprises a tri-hydrochloride salt of the compound.
 26. Thepharmaceutical composition of claim 21, wherein the pharmaceuticalcomposition is substantially free of polymorphs of Form B, Form C, FormD, and Form E.
 27. The pharmaceutical composition of claim 21, whereinthe compound has a DSC thermogram substantially in accordance with FIG.5 .
 28. A pharmaceutical composition comprising a Form A polymorph of acompound of Formula (X):

wherein the Form A polymorph exhibits a dehydration at about 25.9° C. toabout 150.0° C. with a weight loss of about 3.46% in a thermogravimetricanalysis (TGA).
 29. The pharmaceutical composition of claim 28, whereinthe pharmaceutical composition comprises a hydrochloride salt of thecompound.
 30. The pharmaceutical composition of claim 29, wherein thepharmaceutical composition comprises a hydrated form of the compound.31. The pharmaceutical composition of claim 30, wherein the hydratedform of the compound is a monohydrate.
 32. The pharmaceuticalcomposition of claim 29, wherein the pharmaceutical compositioncomprises a tri-hydrochloride salt of the compound.
 33. Thepharmaceutical composition of claim 28, wherein the pharmaceuticalcomposition is substantially free of polymorphs of Form B, Form C, FormD, and Form E.
 34. A pharmaceutical composition comprising a Form Apolymorph of a compound of Formula (X):

wherein the Form A polymorph has a TGA thermogram substantially inaccordance with FIG. 5 .
 35. The pharmaceutical composition of claim 34,wherein the pharmaceutical composition comprises a hydrochloride salt ofthe compound.
 36. The pharmaceutical composition of claim 35, whereinthe pharmaceutical composition comprises a hydrated form of thecompound.
 37. The pharmaceutical composition of claim 36, wherein thehydrated form of the compound is a monohydrate.
 38. The pharmaceuticalcomposition of claim 35, wherein the pharmaceutical compositioncomprises a tri-hydrochloride salt of the compound.
 39. Thepharmaceutical composition of claim 34, wherein the pharmaceuticalcomposition comprises the composition is substantially free ofpolymorphs of Form B, Form C, Form D, and Form E.
 40. A pharmaceuticalcomposition comprising a Form A polymorph of a compound of Formula (X):

wherein the Form A polymorph has a DSC thermogram substantially inaccordance with FIG. 5 .
 41. The pharmaceutical composition of claim 40,wherein the pharmaceutical composition comprises a hydrochloride salt ofthe compound.
 42. The pharmaceutical composition of claim 41, whereinthe pharmaceutical composition comprises a hydrated form of thecompound.
 43. The pharmaceutical composition of claim 42, wherein thehydrated form of the compound is a monohydrate.
 44. The pharmaceuticalcomposition of claim 41, wherein the pharmaceutical compositioncomprises a tri-hydrochloride salt of the compound.
 45. Thepharmaceutical composition of claim 41, wherein the pharmaceuticalcomposition is substantially free of polymorphs of Form B, Form C, FormD, and Form E.